The overall goal of this proposal is to understand the molecular mechanism underlying fluid and electrolyte secretion by the submandibular salivary gland (SMG) with a focus on the ductal component of the secretion. Vectorial ion transport in salivary epithelial cells requires polarized expression of specific ion transporters. Further, the regulation and localization of the ion transport systems of the duct have been poorly characterized. A comprehensive cell physiological, biochemical, and molecular approach has been proposed to test the model, which is the working hypothesis of this proposal. The aims of the proposal are to: (1) study the role of the newly discovered CFTR and ENaC on HCO3-, Cl- and Na+ efflux in the luminal membrane (LM) of acinar cells.; (2) to examine the "pull-push" model in parallel with a luminal Cl-/HCO3 exchange in mediating transductal Cl-transport; (3) to determine the major mechanism of HCO3 influx in the basolateral membrane (BLM) and K+-HCO3 efflux in the LM; (4) to evaluate whether direct interaction between ENaC and CFTR regulates the activity of the two channels and thereby NaCl absorption and the coupled KHCO3 secretion; and (5) to follow the activity of the individual transport/pathways in the context of the overall model. The study will use methodology previously applied by this group to study ion transporters in pancreatic cells and also more recent techniques established to study the ion transporters in the submandibular gland duct. By using electrophysiological studies (patch clamp measurements), fluorescence measurement (using specific dyes to determine cytosolic pH and the concentrations of various ions), cell volume measurements, and immunocytochemistry, the functional localization of the specific ion transporters in the SMG ductal cell has been proposed.